Enhanced cadmium binding ability in response to novel modifications in a Paramecium cadmium metallothionein PMCd1

Abstract

Metallothioneins (MTs) are low molecular weight cysteine rich proteins involved in detoxification of heavy metals. They are synthesized in response to metal exposure and can bind to various metals, thus reducing their toxicity and providing protection against oxidative stress. MTs are considered to be efficient bioremediators of heavy metal contaminated industrial wastewater. The present study was aimed at further enhancing the metal binding capacity of a cadmium metallothionein protein PMCd1, reported some time back from this laboratory in protozoan ciliate Paramecium, to equip them with more efficient system to deal with metal contaminated water bodies. Three additional cysteine residues were introduced at three different places of the protein by site directed mutagenesis, viz. S20C, R180C and Y185C. The wild type and each mutant of PMCd1 were expressed in E. coli BL21 cells. Metal uptake ability of each transformant was determined in the presence of 1 and 2mM Cd2+ in the medium. The three mutants showed enhanced metal uptake compared to the wild PMCd1 which underscored the role of additional cysteines in enhanced metal binding ability. Amongst the mutants, the genetically modified organism with S20C mutation exhibited 9.1 fold more metal uptake compared to the control ciliate. This mutant has great potential to clean the cadmium- contaminated water.

Fig 1. Wild PMCd1 protein.

(A) Amino acid sequence of the protein. (B) The sequence arranged in repeats X_mCX₂CX₃CX₂CX_nCX₂C (m = 8-15, n = 5-10). Blue boxes show the positions where an X amino acid is replaced by cysteine.

Fig 2. Prediction of potential Cd²⁺-binding pockets in PMCd1.

(A) Surface model of metallothionein with its potential Cd²⁺-binding pockets. (B) Sequences of potential Cd²⁺-binding pockets. (C) 3D structure of wild metallothionein protein. Ser20, Arg180 and Tyr185 are represented as sticks. (D) Superimposition of wild metallothionein and its mutants (S20C-cyan, R180C-orange and Y185C-yellow).

Fig 3. SDS-PAGE showing expression of recombinant proteins: PMCd1 and its mutants.

(A) Protein profiles of whole cell lysates of uninduced and IPTG induced PMCd1 wild and its three mutant transformants. 22.6KDa bands present only in induced samples are respective metallothioneins. (B) Protein profile of whole cell lysate, soluble proteins and insoluble proteins. Wild and all mutant forms expressed mainly in soluble form. L: Protein ladder (Benchmark cat.no. 10747-012), T: Total proteins in cell lysate, S: Supernatant fractions containing soluble proteins, Is: Pellet fraction containing insoluble proteins.

Fig 4. Recombinant plasmids of PMCd1 and its mutants. pET 21a Recombinant plasmids containing wild (PMCd1) and three mutant forms (S20C, R180C and Y185C) were isolated and their integrity was checked on 1% agarose gel.

Single restrictions with Hind III and double restrictions with Nde I and Hind III were performed to confirm the presence of right sized insert in cloning vector pUC57 and in expression vector pET21a. Colony PCR was also performed to further confirm the presence of wild PMCd1 and its three mutant forms in pET21a. 1kb DNA ladder (Fermentas SM0331) was used in each gel.

Fig 5. Effect of different concentrations of cadmium on growth of BL21 cells over a period of 24 h.

Cells grown in the presence of 0-5 mM Cd²⁺ were serially diluted and plated on LB agar plates.

Fig 6. Effect of different concentrations of cadmium on growth of BL21 cells in the liquid culture medium.

Number of cells (log10/ml culture) was counted and plotted to determine non-lethal, sub-lethal and near-lethal ranges of Cd²⁺ for BL21 cells.

Fig 7. Cadmium uptake (in µg/mg dry cell weight) by wild PMCd1 and mutants (R180C, Y185C and S20C) over a period of time showing more uptake at 1mM concentration of Cd.

Fig 8. Comparison of Cd binding capacity of wild type and the three mutants.

(A) Significant differences (p < 0.05) in uptake as compared to respective control in wild PMCd1 are represented by asterisks. (B) Fold increase in Cd²⁺ binding ability in response to each mutation as compared to wild PMCd1 is given in front of respective bar.

Fig 9. Docking of Cd²⁺ into 3 binding pockets of wild type metallothionein.

Docking of Cd²⁺ and other cations into the pocket_1 of metallothionein. Like Cd²⁺, in case of monovalent cation Na⁺ and divalent cations Ca²⁺, Hg²⁺ and Zn^2+, Asp35, Ala41 and Thr43 form direct interactions with the metal ion while Lys36, Asn38 and Val42 are involved in hydrophobic interactions with the metal ion. Amino acids with blue boundaries are alkaline in nature while amino acids with red boundary are acidic. Amino acids represented with green background are hydrophobic in nature. Docking of Cu²⁺ into the pocket_1 of metallothionein showed that Asp35, Ala41 and Thr43 formed direct interactions with the metal ion without retaining the charge of 2+ on Cu. Lys36, Asn38 and Val42 were involved in hydrophobic interactions with the metal ion.

Fig 10. Structural motifs study after site directed mutagenesis in PMCd1.

Presence of CXXCC motif in S20C (A) resulted in maximum enhanced Cd²⁺ binding ability as compared to other two CXXC and CXCXC in R180C (B) and Y185C (C) mutants, respectively. Additional Cys residue in each mutant is highlighted with blue color.